In the fight against COVID, we turn to an old
proverb: When the going gets tough,
the tough solve structures.

We want to start our April newsletter by sending all of you our best wishes and we hope you all are staying safe and healthy during this unprecedented time. Highlighted in this month’s newsletter is important recent structural biology work being done on the SARS-CoV-2 proteins.  
 
The SARS-CoV-2 Main Protease:
Main Protease StructureDue to its essential role in proteolytic processing of replicase polyproteins translated from viral RNA, the main protease is a potential target for therapeutics. In the weeks following the first reported case of COVID-19, a group of researchers led by Haitao Yang, Zihe Rao, and Hualiang Jiang at Shanghai Tech University in China deposited the structure of the SARS-CoV-2 main protease in complex with an inhibitor (PDB: 6LU7)(1). Rolf Hilgenfeld and collaborators at the University of Lubeck also recently published the crystal structure of this protein with and without bound inhibitor (PDB: 6Y2E and 6Y2F)(2), and additionally tested this inhibitor in vitro and in vivo, demonstrating its effectiveness at inhibiting the main protease. Building upon this work, a team at Diamond Light Source are utilizing their X-Chem fragment screening platform to determine crystal structures of the SARS-CoV-2 main protease bound to a panel of different active site fragments(3). As of April 22, 2020, there are over 100 structures of the SARS-CoV-2 main protease deposited in the Protein Data Bank, which are paving the way for the design of novel inhibitors of this protein. For more information on the importance of the main protease, visit the February 2020 PDB Molecule of the Month page.

PDB: 6M17The SARS-CoV-2 Spike Protein:
The spike protein of viruses is necessary for viral entry into host cells. Entry occurs through interaction of the spike protein with the cell receptor ACE2. Due to this role in vial entry, the spike protein is a main target for neutralizing antibodies against the virus. Research groups at the University of Texas at Austin (PDB: 6VSB) and the University of Washington (PDB: 6VXX) have determined the first Cryo-EM structures of the trimeric SARS-CoV-2 spike glycoprotein(4, 5). Additionally, two other groups led by Jinghua Yan and Jianxun Qi at the Chinese Academy of Sciences (PDB: 6LZG), and Xinquan Wang at Tsinghua University (PDB: 6M0J) have determined structures of the receptor-binding domain of the spike protein with human ACE2(6, 7). In related work, Qiang Zhou and coworkers at Westlake University in China published the Cryo-EM structure of the Angiotensin-converting enzyme 2 (ACE2) receptor / neutral amino acid transporter B0AT1 bound to the receptor binding domain of the surface spike protein of SARS-CoV-2 (PDB: 6M17)(8). ACE2 is located on the extracellular surface of cells and serves as an entry point for the SARS-CoV-2 virus through binding of the viral spike protein. To illustrate how these structures of the spike protein and the ACE2 – spike protein complex are aiding in the development of small molecules and/or neutralizing antibodies, the labs of Ian Wilson at Scripps and Chris K. P. Mok at the University of Hong Kong determined the crystal structure of a neutralizing antibody in complex with the receptor binding domain of the SARS-CoV-2 spike protein(9).

Other SARS-CoV-2 Proteins:
Expanding their work on the main protease, the research group led by Zihe Rao also deposited the Cryo-EM structure of the SARS-CoV-2 RNA-dependent RNA polymerase Nsp12/RdRp in complex with cofactors Nsp7 and Nsp8 (PDB: 6M71)(10). This protein catalyzes the synthesis of viral RNA, and is the hypothesized target for the promising antiviral drug, remdesivir. Andrzej Joachimiak and collaborators from the University of Chicago and Argonne National Lab deposited the crystal structure of endoribonuclease Nsp15/NendoU from SARS-CoV-2 (PDB: 6VWW)(11). This protein plays a role in RNA processing, is essential to coronavirus biology, and is a target for potential inhibitors.
 
These stories are a small snapshot in to the unbelievable amount of structural biology work that is being done on these proteins. It is incredible to see how rapidly these structures are being determined and deposited. For a complete and up to date list of all of the COVID19 related protein structures deposited in the Protein Data Bank to date, click here.

Our crystallization screens and detergents:
For the SARS-CoV-2 main protease structure determined by the Hilgenfeld group, initial crystallization hits were obtained in the Molecular Dimensions PACT Premier and Morpheus crystallization screens. Initial crystallization hits of structures determined by the team at Diamond Light Source were obtained using the Molecular Dimensions Proplex crystallization screen. For the work on the SARS-CoV-2 Nsp15/NendoU protein initial crystals were obtained using the Microlytic MCSG 4 crystallization screen. Crystals of the neutralizing antibody bound to the receptor binding domain of the spike protein were obtained using the NeXtal JCSG Core Suite. To determine the Cryo-EM structure of the ACE2 / B0AT1 complex, membrane fractions expressing the protein complex were solubilized in 1% GDN, followed by purification using 0.02% GDN.
 
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 References:
  1. Jin, Z, et al. 2020. Structure of Mpro from COVID-19 virus and discovery of its inhibitors. Nature. doi: 10.1038/s41586-020-2223-y.
  2. Zhang, L, et al. 2020. Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors. Science. doi: 10.1126/science.abb3405
  3. Main protease structure and XChem fragment screen. Diamond Light Source. Accessed: April 6, 2020.
  4. Wrapp, D, et al. 2020. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science. doi: 10.1126/science.abb2507.
  5. Walls, AC, et al. 2020. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell. doi: 10.1016/j.cell.2020.02.058.
  6. Wang, Q, et al. 2020. Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2. Cell. doi: 10.1016/j.cell.2020.03.045.
  7. Lan, J, et al. 2020. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature. doi: 10.1038/s41586-020-2180-5.
  8. Yan, R, et al. 2020. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science. doi: 10.1126/science.abb2762.
  9. Yuan, M, et al. 2020. A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-CoV. Science. doi: 10.1126/science.abb7269.
  10. Gao, Y, et al. 2020. Structure of RNA-dependent RNA polymerase from COVID-19 virus. Science. doi: 10.1126/science.abb7498.
  11. Kim, Y, et al. 2020. Crystal structure of Nsp15 endoribonuclease NendoU from SARS-CoV-2. Protein Sci. doi: 10.1002/pro.3873.